Crystalline cytidine-5{40 -diphosphate choline monohydrate and production thereof

ABSTRACT

Crystalline cytidine-5&#39;&#39;-diphosphate choline monohydrate is produced by A. ADDING A HYDROPHILIC ORGANIC SOLVENT TO AN AQUEOUS SOLUTION SYSTEM OF CYTIDINE-5&#39;&#39;-DIPHOSPHATE CHOLINE IN THE PH range of from about 2 to about 4, or B. KEEPING CYTIDINE-5&#39;&#39;-DIPHOSPHATE CHOLINE STANDING AT A RELATIVE HUMIDITY OF NOT LESS THAN ABOUT 32 PERCENT AND AT A TEMPERATURE IN THE RANGE OF FROM ABOUT 17*C to about 70*C for not shorter than about 5 days.

United States Patent Nakamachi et al.

[451 Aug. 29, 1972 [54] CRYSTALLINE CYTIDINE-S DIPHOSPHATE CHOLINE MONOHYDRATE AND PRODUCTION THEREOF [72] Inventors: Hideo Nakamachi; Kazuhide Kamiya, both of Osaka; Masao Nishikawa; Shoichiro Fuiii, both of Kyoto, all of Japan [73] Assignee: Takeda Chemical Industries, Ltd.,

Osaka, Japan [22 Filed: Aprll24,1970

[21] Appl. No.: 31,547

[30] Foreign Application Priority Data April 24, 1969 Japan ..44/32072 [52] US. Cl. ..260/211.5 R, 260/999 [51] Int. Cl. ..C07d 51/52 [58] Field of Search ..260/21 1.5 R

Primary Examiner-Lewis Gotts Assistant Examiner-Johnnie R. Brown Att0rney-Wenderoth, Lind & Ponack ABSTRACT Crystalline cytidine-S -diphosphate monohydrate is produced by choline a. adding a hydrophilic organic solvent to an aqueous solution system of cytidine-5'-diphosphate choline in the pH range of from about 2 to about 4, or

b. keeping cytidine-5'-diphosphate choline standing at a relative humidity of not less than about 32 percent,

and at a temperature in the range of from about 17C to about 70C for not shorter than about 5 days.

1 Claim, 9 Drawing Figures memsnwszs m2 3.687.932

SHEEI' 1 w 5 Fig- 1;

Fig2- mam/V702) HIDE O NAKAMACHI KAZUHIDE KAMIYA,

MASAO NISHIKAWA, and

WWW SHQICHIRO FUJII I PATENTEDmczs 1912 3.687.932

saw u or 5 HIDEO NAKAMACHI, KAZUHIDE KAMIYA, I MAS AO NISHIKAWA &

's'Ho'IcHIRo FUJII,

PATENIEDwm I912 3 687.932

SHEET 5 OF 5 Fiy-8- MOISTURE CONTENT RELATIVE HUMIDlTY m) HIDEO NAKAMACHI, KAZUHIDE KAMIYA, MAsAo' NISHIKAWA, and SHOICHIRO FUJII,

BYIJMIMMXLMM A ram/5 V5 CRYSTALLINE CYTIDINE-S '-DIPHOSPHATE CHOLINE MONOHYDRATE AND PRODUCTION THEREOF can be shown by the following amphoteric formula:

o on, H I N CHz0-iO-i0OH2-CHzN CHa H ii @i and is useful, for example, as an agent for the treatment of the disturbance of consciousness or neuro-psychiatic symptoms accompanying head injuries and cerebral operations.

GDP-Choline is very readily soluble in water and has such slight solubility gradients to temperature in various organic solvent that, by the known method, the compound has not been crystallized but has merely been obtained in the form of either an oil or an amorphous powder. Therefore, it has heretofore been common practice to recover the compound by lyophilization from the reaction mixture. However, the lyophilized CDP-Choline thus obtained still has an amorphous structure and is accompanied with many disadvantages which make it unsatisfactory in practical use. Those disadvantages are:

l. The lyophilization process is time-consuming and costly;

2. The moisture content of the preparation is not constant.

3. The preparation is difficultly handled owing to its small bulk density and high hygroscopicity; and

4. The preparation is vulnerable to contamination with impurities, especially with pyrogens.

The present inventors have unexpectedly found that when CDP-Choline obtained by the conventional methods is further subjected to specified conditions as detailed hereinafter, novel cytidine-5'-diphosphate choline monohydrate (hereinafter referred to as CDP- Choline monohydrate) is produced as stable crystals in a good yield and that thus-obtained crystalline CDP- Choline monohydrate is not only attractive to look at but also has extremely excellent qualities for practical handling and use.

Thus, the principal object of the present invention is to provide novel crystalline CDP-Choline monohydrate with excellent properties for practical use.

Another object of the present invention is to provide an industrially feasible method for producing the said crystalline CDP-Choline monohydrate.

Crystalline CDP-Choline monohydrate of the invention is produced by subjecting CDP-Choline to further specified crystallization procedure. The industrially feasible crystallization method comprises adding a hydrophilic organic solvent to an aqueous solution system of GDP-Choline. H

The aqueous solution system of CDP-Choline may be those which are prepared by dissolving GDP-Choline in any of the amorphous forms, e.g. lyophilized preparation or oily form, into water, or those obtained in the per se known syntheses procedure of CDP-Choline. When the concentration of GDP-Choline in the aqueous solution is higher than about 50 to 60 percent by weight relative to the aqueous solution system, there is found a tendency toward the precipitation of CDP- Choline itself as an oily substance along with crystals of CDP-Choline monohydrate thus lowering the yield of crystalline GDP-Choline monohydrate. When the concentration of CDP-Choline is not higher than about 20 percent by weight, the yield of crystalline CDP-Choline monohydrate is relatively low. Therefore, the advantageous concentration of GDP-Choline in the aq ueous solution system is higher than about 20 percent but lower than about 60 percent by weight.

It is necessary to previously adjust the aqueous solution system of CDP-Choline to a pH value in the range of from pH about 2 to pH about 4. When the aqueous solution system is more strongly acidic, the solubility of CDP-Choline monohydrate is increased to lower the yield of the objective crystalline CDP-Choline monohydrate. On the other hand, in the pH range above about 4, there is formed the corresponding alkali salt of CDP-Choline which decreased the yield of the objective crystalline CDP-Choline monohydrate.

The aqueous solution system of CDP-Choline may contain a hydrophilic organic solvent in a small amount which is sufficient to avoid precipitation of CDP- Choline as the raw material.

As the hydrophilic organic solvent, use may be made of various organic solvents miscible with water, such as methyl alcohol, ethyl alcohol, acetone, dioxane, dimethyl sulfoxide, an optional mixture thereof and the like. Among those hydrophilic organic solvents, some solvents such as methyl alcohol and dimethyl sulfoxide are particularly conducive to the rapid crystallization of CDP-Choline monohydrate and other solvents such as acetone and dioxane are excellent in giving a high yield of the objective crystalline CDP-Choline monohydrate. It is therefore especially advantageous to firstly add the former e.g. methyl alcohol and/or dimethyl sulfoxide to the aqueous solution system of CDP-Choline to promote the crystallization of CDP- Choline monohydrate, and then, to add the latter e.g. acetone and/or dioxane to provide for an increased yield of crystalline CDP-Choline monohydrate.

When ethyl alcohol is employed as the hydrophilic organic solvent, the formation of crystalline CDP- Choline monohydrate may be promoted by the addition of an aqueous solution of ethyl alcohol in the concentration of about percent, e.g. in the range of about 70 to percent.

Though the amount of the hydrophilic organic solvent to be added into the aqueous solution system of GDP-Choline may vary somewhat with the kinds of the hydrophilic organic solvents and whether or not the aqueous solution system previously contains a hydrophilic organic solvent, it is generally sufficient to add the hydrophilic organic solvent in an amount to make its total amount about 5 to about 20 times the volume of water contained in the aqueous solution system. Usually, it is a good expedient to add a hydrophilic organic solvent, after no more precipitates form in the resulting mixture, in a further amount corresponding to about 30 percent of that so far contained in the aqueous solution system.

From the viewpoint of the yield and purity of crystalline GDP-Choline monohydrate, it is generally advantageous to add the hydrophilic organic solvent dropwise to the aqueous solution system of CDP- Choline under continuous or intermittent stirring.

The formation of crystalline CDP-Choline monohydrate is accelerated when said addition is carried out at an elevated temperature, but use of a too high temperature will result in thermal decomposition of GDP-Choline. Meanwhile, if the solvent addition is conducted at an excessively low temperature, the formation of crystalline CDP-Choline monohydrate requires a prolonged period of time. Generally speaking, it is advantageous to add a hydrophilic organic solvent at a temperature in the range of about 20C to about 70C.

It is possible to allow CDP-Choline monohydrate to crystallize out in an extremely shortened period of time by adding previously prepared crystalline CDP-Choline monohydrate as seed crystals especially when the resulting mixture shows signs of turbidity in the process of adding a hydrophilic organic solvent to an aqueous solution system of GDP-Choline.

In a further embodiment of the invention, crystalline CDP-Choline monohydrate is produced by keeping the amorphous CDP-Choline, e.g. the lyophilized CDP- Choline, standing at a relative humidity of not less than about 32 percent and at a temperature in the range of from about 17 to about 70C. In this case, the amorphous CDP-Choline absorbs a substantial amount of water and is dissolved in the moisture to form a liquid state, but further keeping the same standing under the above-mentioned conditions yields crystalline CDP-Choline monohydrate with the evaporation of an excess of the absorbed water.

The speed of the formation of crystalline CDP- Choline monohydrate increases with elevated temperature, but at a temperature above about 70C the speed is too high to allow the monohydration to proceed into the inside of the amorphous CDP-Choline. Thus, a mere skin of the monohydrate is formed only in the surface layer of the amorphous CDP-Choline. In addition, the thermal decomposition of CDP-Choline is also promoted to detract from the yield of crystalline CDP- Choline monohydrate. A temperature lower than about 17C is not advantageous since CDP-Choline monohydrate is formed too slowly.

Practically, it is most advantageous from the viewpoint of the crystallization speed, yield and purity of the crystalline CDP-Choline monohydrate to keep amorphous CDP-Choline standing under the conditions of temperature and relative humidity which, as plotted against each other on rectangular co-ordinate system, lie within the quadrilateral area defined by the four apices (17C, 98 percent), (17C, 56 percent), (70C, 66 percent) and (70C, 32 percent).

The time required for the formation of crystalline CDP Choline monohydrate varies somewhat with the temperature, relative humidity and other conditions,

but usually a period of not shorter than about 5 days is required. In addition, the formation speed of crystalline CDP-Choline monohydrate may be increased by adding previously prepared crystalline CDP-Choline .monohydrate as seed crystals.

FIG. 1 of the accompanying drawing is a reproduction of a photograph of crystals of CDP-Choline monohydrate of the present invention magnified 40 diameters with a microscope.

CDP-Choline monohydrate shows a well defined X ray difiraction pattern which indicates that this compound is highly crystalline. The X-ray diffraction pattern of CDP-Choline monohydrate measured by the powder method is as shown in FIG. 2 of the accompanying drawing, and the significant lattice spacings are as follows:

2.7 angstrom (middle) 3.0 angstrom (middle) 3.1 angstrom (weak) 3.3 angstrom (middle) 3.55 angstrom (middle) 3.75 angstrom (doublet, middle) 4.0 angstrom (very strong) 4.3 angstrom (strong) 4.6 angstrom (middle) 4.75 angstrom (middle) 5.1 angstrom (middle) 5.8 angstrom (middle) 6.3 angstrom (middle) 8.1 angstrom (middle) 1 1.5 angstrom (weak) Lyophilized preparation of CDP-Choline shows no significant X-ray diffraction pattern by the powder method as clearly seen in FIG. 3 of the accompanying drawing and therefore it has none of the above-listed lattice spacings. Thus, crystalline CDP-Choline monohydrate of the present invention is essentially characterized by the above-listed lattice spacings.

Further physico-chemical properties of the crystalline CDP-Choline monohydrate are as follows:

Infrared absorption spectrum measured by the Nujol mull method is as shown in FIG. 4 of the accompanying drawing, and the significant absorptions in wave length are as follows:

3.0, 5.8, 6.0, 6.3, 6.55, 7.8, 8.1, 8.3(broad, doublet), 8.9, 9.25, 9.4, 9.7, 10.1, 10.4, 10.6, 11.05, 11.5(broad), 11.9,12.3, 12.8 and 14.0 microns.

The infrared absorption spectrum of crystalline CDP- Choline monohydrate is clearly different from that of amorphous CDP-Choline which is shown in FIG. 5 of the accompanying drawing (the Nujol mull method).

The change in weight of crystalline CDP-Choline monohydrate determined by means of the thermogravimetric analysis is as shown in FIG. 6 of the accompanying drawing, which clearly demonstrates that it contains 1 mole water of crystallization.

Crystallographic data:

Colorless Space-group; P2 2 2 Unit-cell dimensions;

a-axis l 1.&0.2 angstrom b-axis 22.8103 angstrom c-axis 8.6101 angstrom Number of molecules in a unit-cell; 4 FIG. 7 of the accompanying drawing is to illustrate the stereo model of CDP-Choline molecule in the crystal of CDP-Choline monohydrate projected in direction of the c-axis.

Crystalline CDP-Choline monohydrate of the present invention offers the following advantages over the conventional preparations of CDP-Choline.

l High stability against moisture:

In FIG. 8 of the accompanying drawing, the lines designated A, B, C and D show, respectively, the moisture absorption equilibrium curve of a lyophilized preparation of CDP-Choline as measured at C, that of the said preparation as measured at 40C, that of crystalline CDP-Choline monohydrate as measured at 40C. As is clearly seen in FIG. 8, crystalline CDP- Choline monohydrate remains stable up to the relative humidity of 75 percent even at 40C, and at the temperature of 20C, shows no change even when the relative humidity is as high as 95 percent. In sharp contrast thereto, the lyophilized preparation of CDP-Choline is highly hygroscopic and liable to deliquescence. In addition, this hygroscopicity gains with increasing temperatures. Therefore, the lyophilized preparation of CDP- Choline not only requires the utmost care in its production but lends to itself only poorly to the uniformity of quality. This is sharp contrast of crystalline CDP- Choline monohydrate which, even if not stored in an air-tight or closed container, does not undergo a change of any appreciable degree in quality, and therefore, which requires no special care in handling.

2. Superior thermal behaviors:

The melting point (decomposition point) of the lyophilized CDP-Choline varies considerably with differences in moisture content, and even a freshly prepared sample melts (decomposes) gradually between 190C and 196C (uncorrected). which is higher than said range by at least about 30C. Furthermore, crystalline CDP-Choline monohydrate is more stable against heat than the lyophilized preparation of CDP-Choline.

For example, the following table shows the respective colorations in terms of reflectivity at 360 my. of the said two compounds on heating at 80C for 70 hours.

Further, the lyophilized preparation of CDP-Choline is obtained in various moisture contents and, as shown in FIG. 9 of the accompanying drawing, its weight change starts at about 30C and continues to about 140C. In contrast, crystalline CDP-Choline monohydrate retains its water of crystallization up to higher than about 100C and it abruptly loses its water of crystallization at about 110 to about 120C becoming an anhydrate as shown in FIG. 6 of the accompanying drawing. Thus, crystalline CDP-Choline monohydrate does not suffer changes in amount of its water of crystallization by the general differences in drying conditions and, therefore, lends itself better to the maintenance of product uniformity.

3. It has a stable crystalline structure:

The anhydrate obtained by heating crystalline CDP- Choline monohydrate to a temperature higher than about 120C has a crystalline structure different from that of CDP-Choline monohydrate. When this crystalline anhydrate is kept standing overnight at 20C and at about 60 percent of relative humidity, it regains water of crystallization to form the original crystalline CDP- Choline monohydrate.

This fact is clearly indicative of the stable crystalline structure of CDP-Choline monohydrate.

4. Ease in handling;

Lyophilized preparation of CDP-Choline is not only hygroscopic as described about but has a low bulk density and a low fluidity.ln sharp contrast thereto, CDP- Choline monohydrate comes in the form of attractive crystals which have remarkably higher bulk density and a greater fluidity than the conventional preparations.

5. Substantial freedom from contamination;

Lyophilized preparation of CDP-Choline inevitably contains impurities, especially pyrogens, because only the solvent is evaporated in the lyophilization process and all the non-volatile materials in the solution find their way into the preparation as impurities. Even CDP- Choline recovered by coagulating oily CDP-Choline .from a concentratedsolution is liable to get contaminated with foreign materials which have been dissolved in the oily CDP-Choline. In contrast, CDP- Choline monohydrate of the present invention is precipitated from its solution as fine crystals and, as such, is free from contamination with impurities, especially with pyrogens.

6. Reductions in procedural complexity and production cost:

Crystalline CDP-Choline monohydrate is obtained by the simple procedure and low cost according to the method of the present invention in which the lyophilization process is not necessary.

Crystalline CDP-Choline monohydrate of the present invention shows thesame pharmacological activities as those of the conventional lyophilized preparation of CDP-Choline and is used as the agent for the treatment of the disturbance of consciousness or neuro-psychiatric symptoms accompanying head injuries and cerebral operations, and the like. Generally, about 100 to about 500 milligrams is administered once or twice a day by intravenous drip, intravenous injection or intramuscular injection. A dose of about 250 to about 500 milligrams is most effective.

The following examples are merely for illustrative purposes and are not to be construed as the limitation of the present invention. Throughout the present specification, the abbreviations mg, ml, and C refer respectively to milligram(s), milliliter(s) and degrees centigrade, parts by volume bear the same relationship to parts by weight as do milliliters to grams and percentage are volume per volume except the case where the meaning is clearly otherwise from the context.

EXAMPLE I To a solution of 9.5 parts by weight (on dry basis) of CDP-Choline in 20 parts by volume of water is added 20 parts by volume of ethyl alcohol at C. While the mixture is stirred at the same temperature, 40 parts by volume of percent ethyl alcohol is added thereto dropwise over 2 hours. After the addition is completed,

about 20C with stirring for 10 hours to yield crystals of 5 CDP-Choline monohydrate. The crystals are recovered by filtration, washed with 50 parts by volume of ethyl alcohol, and dried in a current of air at about C. Yield: 9.2 parts by weight or 93.4 percent.

EXAMPLE 2 To a solution of 4.8 parts by weight (on dry basis) of CDP-Choline in 5 parts by volume of water is added 10 parts by volume of methyl alcohol at 50C. While the mixture is stirred well at the same temperature, parts by volume of methyl alcohol is gradually added thereto dropwise over 3 hours. After the addition is completed, the mixture is further stirred at the same temperature for 1 hour, then cooled to 45C, followed by the dropwise addition of 20 parts by volume of acetone over 1 hour. The mixture is cooled to about 20C and is kept standing overnight to yield crystals of CDP-Choline monohydrate. The crystals are recovered by filtration, washed with parts by volume of acetone and dried in a current of air at 20C. Yield: 4.6 parts by weight or 92.4 percent.

EXAMPLE 3 To a solution of 9.5 parts by weight (on dry basis) of CDP-Choline in 10 parts by volume of water is added 20 parts by volume of methyl alcohol at 20C. While the mixture is stirred well at the same temperature, 50 parts by volume of methyl alcohol is added thereto dropwise over 1 hour. When the addition of 20 parts by volume of methyl alcohol is completed, 0.05 part by weight of crystalline CDP-Choline monohydrate is EXAMPLE 4 To a solution of 30 parts by weight (on dry basis) of CDP-Choline in 30 parts by volume of water is added 60 parts by volume of methyl alcohol at 60C. While the mixture is stirred well at the same temperature, 150 parts by volume of methyl alcohol is added thereto dropwise over 3 hours. After the addition is completed, the mixture is gradually cooled to about 20C to yield crystals of CDP-Choline monohydrate. The crystals are recovered by filtration, washed with 10 parts by volume of methyl alcohol and dried at 40C under reduced pressure.

Yield: 29 parts by weight or 93.2 percent.

EXAMPLE 5 To a solution of parts by weight (on dry basis) of GDP-Choline in 400 parts by volume of water is added 2,500 parts by volume of dimethyl sulfoxide dropwise over 2 hours, whereby crystals of CDP-Choline monohydrate are formed. The crystals are recovered by filtration and dried at 40C under reduced pressure. Yield: 97 parts by weight or 93.5 percent.

EXAMPLE 6 950 mg. (on dry basis) of lyophilized preparation of CDP-Choline is spread on a dish about 5 centimeters in diameter. The dish is then placed in a desiccator having a relative humidit of 62 erc nt controlled with a saturated aqueous so ution 0 so rum nitrite in its bottom.

'The desiccator is allowed to stand in an incubator EXAMPLE 7 950 mg. dry basis) of lyophilized preparation of GDP-Choline is spread on a dish about 5 centimeters in diameter. The dish is then placed in a desiccator having a relative humidity of 84 percent controlled with a saturated aqueous solution of calcium bromide in its bottom. The desiccator is further positioned in an incubator maintained at 20 i 1C. After a week, the dish is taken out and the crystals of CDP-Choline monohydrate formed on the dish are recovered with a spoon.

Yield: 985 mg. or 100 percent.

Having thus disclosed the invention, what is claimed 1s:

1. Crystalline cytidine-5'-diphosphate choline monohydrate having the following lattice spacings:

2.7 angstrom (middle) 3.0 angstrom (middle) 3.] angstrom (weak) 3.3 angstrom (middle) 3.55 angstrom (middle) 3.75 angstrom (doublet, middle) 4.0 angstrom (very strong) 4.3 angstrom (strong) 4.6 angstrom (middle) 4.75 angstrom (middle) 5.] angstrom (middle) 5.8 angstrom (middle) 6.3 angstrom (middle) 8.1 angstrom (middle) 1 1.5 angstrom (weak) 

